B-cell Antigen Receptor Research Articles

B-cell antigen receptor expression and phosphatidylinositol 3-kinase signaling regulate genesis and maintenance of mouse chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a frequent lymphoproliferative disorder of B cells. Although inhibitors targeting signal proteins involved in B-cell antigen receptor (BCR) signaling constitute an important part of the current therapeutic protocols for CLL patients, the exact role of BCR signaling, as compared to genetic aberration, in the development and progression of CLL is controversial. In order to investigate whether BCR expression per se is pivotal for the development and maintenance of CLL B cells, we used the TCL1 mouse model. By ablating the BCR in CLL cells from TCL1 transgenic mice, we show that CLL cells cannot survive without BCR signaling and are lost within 8 weeks in diseased mice. Furthermore, we tested whether mutations augmenting B-cell signaling influence the course of CLL development and its severity. The phosphatidylinositol-3-kinase (PI3K) signaling pathway is an integral part of the BCR signaling machinery and its activity is indispensable for B-cell survival. It is negatively regulated by the lipid phosphatase PTEN, whose loss mimics PI3K pathway activation. Herein, we show that PTEN has a key regulatory function in the development of CLL, as deletion of the Pten gene resulted in greatly accelerated onset of the disease. By contrast, deletion of the gene TP53, which encodes the tumor suppressor p53 and is highly mutated in CLL, did not accelerate disease development, confirming that development of CLL was specifically triggered by augmented PI3K activity through loss of PTEN and suggesting that CLL driver consequences most likely affect BCR signaling. Moreover, we could show that in human CLL patient samples, 64% and 81% of CLL patients with a mutated and unmutated IgH VH, respectively, show downregulated PTEN protein expression in CLL B cells if compared to healthy donor B cells. Importantly, we found that B cells derived from CLL patients had higher expression levels of the miRNA-21 and miRNA-29, which suppresses PTEN translation, compared to healthy donors. The high levels of miRNA-29 might be induced by increased PAX5 expression of the B-CLL cells. We hypothesize that downregulation of PTEN by increased expression levels of miR-21, PAX5 and miR-29 could be a novel mechanism of CLL tumorigenesis that is not established yet. Together, our study demonstrates the pivotal role for BCR signaling in CLL development and deepens our understanding of the molecular mechanisms underlying the genesis of CLL and for the development of new treatment strategies.

Burgeoning Data on BTK Inactivating Mutations in Lymphomagenesis and Therapeutic Resistance

The B-cell antigen receptor signaling pathway has been a primary focus in the targeted treatment of B-cell malignancies for the past decade. When aberrantly activated, this pathway initiates a cascade of phosphorylation mediated by several tyrosine kinases, with Bruton’s tyrosine kinase (BTK) being essential among them. Multiple generations of covalent and non-covalent BTK inhibitors have revolutionized therapeutic options for several B-cell lymphomas. However, the use of continuous BTK inhibition is limited by development of resistance resulting from acquired point mutations that allow persistent B-cell receptor signaling. Genomic sequencing of patient samples at disease progression has recently led to the discovery of novel resistance mutations in BTK that result in diminished or absent BTK kinase activity (termed kinase-deficient). However, the mechanisms underlying the potential advantage of kinase-deficient BTK mutations are incompletely understood and still under investigation. In this review, we provide a background of the pathway leading to the development of current therapies that target BTK and review the literature describing kinase-deficient BTK mutations. We propose that BTK inactivating mutations provide an advantage to neoplastic B-lymphocytes in patients with BTK inhibitor resistant B-cell malignancies and highlight potential mechanisms through which BTK kinase-deficient mutations could be acting, either due to differential protein conformation or by behaving as a scaffold for other signaling molecules. Due to the novelty of these mutations and their increasing rate of incidence over the last decade, it is imperative to continue studying BTK kinase deficient mutations across B-cell malignancies and to propose alternate therapies that could target them.

The Determinants of B Cell Receptor Signaling as Prototype Molecular Biomarkers of Leukemia.

Advanced genome-wide association studies (GWAS) identified several transforming mutations in susceptible loci which are recognized as valuable prognostic markers in chronic lymphocytic leukemia (CLL) and B cell lymphoma (BCL). Alongside, robust genetic manipulations facilitated the generation of preclinical mouse models to validate mutations associated with poor prognosis and refractory B cell malignancies. Taken together, these studies identified new prognostic markers that could achieve characteristics of precision biomarkers for molecular diagnosis. On the contrary, the idea of augmented B cell antigen receptor (BCR) signaling as a transforming cue has somewhat receded despite the efficacy of Btk and Syk inhibitors. Recent studies from several research groups pointed out that acquired mutations in BCR components serve as faithful biomarkers, which become important for precision diagnostics and therapy, due to their relevant role in augmented BCR signaling and CLL pathogenesis. For example, we showed that expression of a single point mutated immunoglobulin light chain (LC) recombined through the variable gene segment IGLV3-21, named IGLV3-21R110, marks severe CLL cases. In this perspective, we summarize the molecular mechanisms fine-tuning B cell transformation, focusing on immunoglobulin point mutations and recurrent mutations in tumor suppressors. We present a stochastic model for gain-of-autonomous BCR signaling and subsequent neoplastic transformation. Of note, additional mutational analyses on immunoglobulin heavy chain (HC) derived from non-subset #2 CLL IGLV3-21R110 cases endorses our perspective. Altogether, we propose a model of malignant transformation in which the augmented BCR signaling creates a conducive platform for the appearance of transforming mutations.

Tracing Human IgE B Cell Antigen Receptor-Bearing Cells With a Monoclonal Anti-Human IgE Antibody That Specifically Recognizes Non-Receptor-Bound IgE.

Up to 30% of the population suffers from immunoglobulin E (IgE)-mediated allergies. Despite current stepwise gating approaches, the unambiguous identification of human IgE-producing cells by flow cytometry and immunohistology remains challenging. This is mainly due to the scarcity of these cells and the fact that IgE is not only expressed in a membrane-bound form on the surface of IgE-producing cells in form of the B cell antigen receptor (BCR), but is more frequently found on various cell types bound to the low and high affinity receptors, CD23 and FcϵRI, respectively. Here we sought to develop a sequential gating strategy for unambiguous detection of cells bearing the IgE BCR on their surface. To that aim we first tested the monoclonal anti-IgE antibody omalizumab for its ability to discriminate between IgE BCR and receptor-bound IgE using cells producing IgE or bearing IgE bound to CD23 as well as basophils exhibiting FcϵRI receptor-bound IgE. Using flow cytometry, we demonstrated that omalizumab recognized IgE producing cells with a high sensitivity of up to 1 IgE+ cell in 1000 human peripheral blood mononuclear cells (PBMCs). These results were confirmed by confocal microscopy both in cell suspensions as well as in nasal polyp tissue sections. Finally, we established a consecutive gating strategy allowing the clear identification of class-switched, allergen-specific IgE+ memory B cells and plasmablasts/plasma cells in human PBMCs. Birch pollen specific IgE+ memory B cells represented on average 0.734% of total CD19+ B cells in allergic patients after allergen exposure. Thus, we developed a new protocol for exclusive staining of non-receptor bound allergen-specific IgE+ B cell subsets in human samples.

A role for FcγRIIB in the development of murine bleomycin-induced fibrosis

BackgroundSystemic sclerosis (SSc) is a systemic autoimmune disease characterized by excessive fibrosis. FcγRIIB is a low-affinity receptor for the Fc fragment of IgG. FcγRIIB is expressed on the surface of various leukocyte subsets and signals negative feedback pathways to down-regulate B-cell antigen receptor signaling. ObjectiveThe aim of the present study was to investigate the role of FcγRIIB in the development of a murine bleomycin-induced scleroderma model. MethodsThe experimental fibrosis model was generated by the intradermal injection of bleomycin into wild-type (WT) and FcγRIIB-deficient (FcγRIIB-/-) mice. We histologically assessed skin and lung fibrosis as well as inflammatory cell infiltration. Cytokine and chemokine expression levels were measured with RT-PCR. ResultsThe severity of fibrosis in the skin and lung was significantly worse in FcγRIIB-/- mice than in WT mice. In the skin of bleomycin-treated mice, the numbers of CD8+ T cells, F4/80+ macrophages, MPO+ neutrophils, NK1.1+NK cells, and B220+ B cells were significantly higher in FcγRIIB-/- mice than in WT mice. The expression of TNF-α and IL-1β was significantly higher in FcγRIIB-/- mice than in WT mice as was the expression of ICAM-1, CXCL2, and CCL3 in the affected skin. An adoptive transfer of splenic leukocytes from FcγRIIB-/- mice into WT mice showed exacerbated skin and lung fibrosis compared to WT mice without an adoptive transfer. ConclusionThese results indicate that FcγRIIB plays an inhibitory role in skin and lung fibrosis. Moreover, modulating FcγRIIB signaling has potential as a therapeutic approach for SSc.

Chromatin Content Capture Reveals Acute Leukaemia Oncogenic Vulnerability Point in Human B Cell Development

Human B cell development in adult human bone marrow (BM) is tightly regulated through well-defined stages to produce adaptive immune cells with assembled and functional B cell antigen receptor (BCR)(Martin et al., 2016). To produce mature B cells with functional immunoglobulin receptors, B cell progenitors must undergo multiple stages of highly regulated chromatin remodelling and transcriptional reprogramming which correspond to unique patterns of surface protein expression (Nutt and Kee, 2007). This complex process is frequently dysregulated in B cell neoplasia such as B cell Acute Lymphoblastic Leukemia (B-ALL). B-ALL is highly heterogenous in its phenotypic and clinical presentation, as well as in its underlying molecular features such as DNA methylation patterns and genetic aberrations (Cobaleda and Sánchez‐García, 2009). The lack of general mechanism of leukemogenesis has made it difficult to identify when and where adult and pediatric B-ALL blasts diverge from normal B cell development. Here we show that across 5 B-ALL patients and 3 cell lines with diverse phenotypic and clinical presentations, blasts are epigenetically arrested at a conserved point within healthy human B cell development.First, we sought to establish a trajectory of normal B cell development to delineate the phenotypic and concomitant epigenetic changes occurring in BM progenitors as they differentiate into naïve B cells. To capture phenotype, function, and epigenetic state via single cell chromatin content (chromotype) of developing B cells in BM, we developed a multiplexed, high throughput, single cell proteomic method (chromotyping) to simultaneously measure cell surface markers, intracellular regulators such as transcription factors and chromatin structure regulators such as histone post-translational modifications (i.e. H3K4me3, H3K27me3, H2AK119ubi) and chromatin re-modelers (i.e. CTCF, DNMT1, MLL1). Using these surrogates for single cell, global chromatin content, we notably identified 3 coordinated epigenetic inflection or switch (S) points in healthy B cell development corresponding to previously characterized phenotypic landmarks of STAT5 signalling and active re-arrangement of IgH loci (S1), CD24 expression-linked high translation and proliferation (S2), and IgM and CD20 expression-linked BCR assembly completion (S3) (Bendall et al., 2014).To determine how these coordinated chromotypes translated to chromatin accessibility and primed gene regulation networks, we isolated BM B cell population from these chromatin content transition points and analysed them with our modified ATAC-seq protocol, InTAC-seq (Baskar et al., 2021). Strikingly, the chromatin accessibility landscape revealed putative oncogenic priming with high activity of leukemic TFs such as PAX5, TCF3, ZEB1 and ID4 predominantly at S2 and some at S3 switch points. By integrating our InTAC-seq data with publicly available single cell ATAC and RNA seq data on BM, we located this oncogenic primed state as existing from S2 to before S3 (IgH rearranged, late pro- / Pre-B cell stage) in healthy B cell development. This integration further associated this state with high activity of ASCL1 (role in chromatin remodelling) and high expression of STMN1 (Leukaemia-associated phosphoprotein 18).Finally we showed that across B-ALL patients (n=5) and cell lines (REH, NALM6, SUBP15), chromatin accessibility of neoplastic B cells indeed continue to occupy this point of oncogenic vulnerability in the B cell developmental space from S2 to right before S3 in our integrated scATAC map, despite variable immunophenotypes. This corresponds to a coordinated minima in our chromotyping map (lowest, coordinated abundance of chromatin structure regulators across trajectory). Further analysis of B-ALL patients reinforced the divergence between immunophenotypic and epigenetic heterogeneity within and between samples. Taken together, our findings identify key epigenetic switch points in B cell development and their underlying chromatin accessibility and gene expression patterns. Consequently, we reveal a point of epigenetic vulnerability in healthy B cell development that could be predisposed to leukemic transformation. This work opens up the possibility for new diagnostic strategies for B-ALL utilizing chromatin content and could pave the way for epigenetic modulation-based treatments beyond DNA methylation inhibition. DisclosuresDavis: Novartis Pharmaceuticals: Honoraria; Jazz Pharmaceuticals: Research Funding.

Single Capture High Throughput Sequencing Assay for Combined V(D)J Clonality Analysis and Oncogene Mutations in the Diagnosis of T and B Lymphoid Malignancies

IntroductionThe diagnosis of B and T cell malignancies relies on the demonstration of B-cell (BCR) or T-cell (TCR) antigen receptor clonality. This can be studied through the analysis of V(D)J rearrangements of BCR and TCR genes by PCR (van Dongen Leukemia 2003) or, more recently, by high-throughput sequencing (HTS). Amplification of a clonal population with a “primers approach” could fail in case of hybridization problems due to too fragmented DNA, somatic mutations or polymorphic variations. Here we evaluated the performance of a HTS capture system for the analysis of B and T-cell clonality in clinical samples from mature T or B malignancies. We further combined this technology to concomitant sequencing of oncogenes of interest.Patients and MethodsDNA was extracted from 58 tumoral samples from fresh/frozen (FF) cells or tissues or formalin-fixed paraffin-embedded tissue (FFPE) (n=19). These samples comprised various T-cell [i.e. 1T-cell prolymphocytic leukemia, 1 T large granular lymphocytic leukemia, 2 Sézary syndrome, 4 peripheral T-cell lymphoma not otherwise specified, 14 angioimmunoblastic T-cell lymphoma] or B-cell [i.e. 14 chronic lymphocytic leukemia, 1 mantle cell lymphoma, 5 diffuse large B-cell lymphoma, 1 grey-zone lymphoma, 13 Hodgkin lymphoma, 1 Poppema, 2 Waldenström and 1 multiple myeloma] malignancies. The Biomed-2 PCR technique was used as standard for assessing the performance of TRG, IGH and IGK clonality analysis. An extensive panel of capture probes was designed (SureSelect XT HS2 DNA system, Agilent Technologies) that covered the variable (V), + diversity (D) and junction (J) segments of the IGH, IGK, TRG, TRB loci and diagnostic/theranostic genes of interest i.e. B2M, BTK, CARD11, CD28, DNMT3A, IDH2, JAK3, PLCG1, PLCG2, ROHA, SOCS1, STAT3, STAT5B, STAT6, TET2, TNFAIP3, TP53. Paired-End sequencing was performed on a MiSeq system (Illumina) in 300, 500 and 600 cycles. Analysis of clonality profiles was performed using Vidjil software and SeqOne.ResultsHTS runs resulted in a median total read count of 1,6M (0.7-2.9) per sample. V(D)J rearrangements were identified with a median of 1503 reads (189-6824) per sample. Five samples were excluded because less than 300 rearranged reads were obtained. The number of rearranged reads and of clonotypes identified are influenced by the number of sequencing cycles (300<500 or 600) but not by the quality of DNA (FFPE vs FF).Analyses of tumoral samples with HTS versus PCR were compared. For the IGH locus (n=47), comparable PCR/HTS clonal (n=22) and polyclonal (PCL, n=20) profiles were identified. One discordant case showed a clonal PCR profile and a PCL HTS profile but the IGK was clonal. For the IGK locus (n=23), 10 clonal and 12 PCL cases were similar with both techniques. One case appeared discordant with a PCL PCR profile but a clonal HTS profile. For the TRG locus (n=31), PCR and HTS profiles were similar in 14 clonal, 5 oligoclonal and 9 PCL cases respectively. Three cases were discordant with oligoclonal PCR profiles but a clonal HTS profile.Overall in the 38 cases of B-cell malignancies, 27 and 11 cases had a concordant B-cell clonal or PCL profile with PCR and HTS. Among PCL cases, only one was discordant with a clonal HTS profile. This case and 3 other PCL cases were Hodgkin lymphomas which all disclosed another mutation (i.e. TP53, TNFAIP3, SOCS1). Of the 20 cases of T-cell malignancies, 14 displayed a clonal TRG profile with PCR and HTS. Among them, 13 showed oncogene mutations that confirmed the oncogenic nature of the clonal proliferation. Among 6 patients with a non-clonal PCR TRG profile, two cases of AITL and T-LGL had a discordant clonal TRG HTS profile and both also had specific mutations (SOCS1, RHOA and STAT3 respectively). Two other AITL samples showed a T-PCL profile with PCR and HTS but also had a mutation/CNV (RHOA, SOCS1).ConclusionA very good performance of B and T cell clonality assessment was obtained here with capture-HTS compared to Biomed-2 PCR. The combined identification of mutation/CNV allowed to confirm the malignant character in cases of clonal or PCL lymphoproliferations, while concomitantly specifying the type of lymphoproliferative disorder. The combined capture-HTS of B and T repertoires and oncogenes of diagnostic or theranostic interest thus appears as an efficient, accurate and useful approach for the diagnosis of mature B and T lymphoid malignancies in clinical practice. DisclosuresNo relevant conflicts of interest to declare.

Acute Csk inhibition hinders B cell activation by constraining the PI3 kinase pathway

T cell antigen receptor (TCR) and B cell antigen receptor (BCR) signaling are initiated and tightly regulated by Src-family kinases (SFKs). SFKs positively regulate TCR signaling in naïve T cells but have both positive and negative regulatory roles in BCR signaling in naïve B cells. The proper regulation of their activities depends on the opposing actions of receptor tyrosine phosphatases CD45 and CD148 and the cytoplasmic tyrosine kinase C-terminal Src kinase Csk. Csk is a major negative regulator of SFKs. Using a PP1-analog-sensitive Csk (CskAS) system, we have previously shown that inhibition of CskAS increases SFK activity, leading to augmentation of responses to weak TCR stimuli in T cells. However, the effects of Csk inhibition in B cells were not known. In this study, we surprisingly found that inhibition of CskAS led to marked inhibition of BCR-stimulated cytoplasmic free calcium increase and Erk activation despite increased SFK activation in B cells, contrasting the effects observed in T cells. Further investigation revealed that acute CskAS inhibition suppressed BCR-mediated phosphatidylinositol 3,4,5-trisphosphate (PIP3) production in B cells. Restoring PIP3 levels in B cells by CD19 cross-linking or SHIP1 deficiency eliminated the negative regulatory effect of CskAS inhibition. This reveals the critical role of Csk in maintaining an appropriate level of SFK activity and regulating PIP3 amounts as a means of compensating for SFK fluctuations to prevent inappropriate B cell activation. This regulatory mechanism controlling PIP3 amounts may also contribute to B cell anergy and self-tolerance.

Analysis of DNA methylation of CD79B in MDV-infected chicken spleen

Marek's disease (MD), an immunosuppressive disease induced by Marek's disease virus (MDV), provides an ideal model for studying diseases caused by a carcinogenic virus. CD79B is a B-cell antigen receptor complex-associated protein β-chain precursor which is involved in the activation, proliferation, differentiation of B-cell and the transmission of downstream signals. This study analyzed CD79B gene mRNA expression and methylation by two schemes #20 (5´ flanking to intron 1) and #27 (intron 2 to intron 3), between MDV-infected tumorous spleens (TS) and non-infected spleens (NS). Results showed that average methylation levels of CpGs in #20 and #27 were higher in TS than in NS (P<0.05), while, CD79B mRNA expression was lower in TS than in NS (P<0.01). Six of 40 CpG sites showed significantly (P<0.05) different methylation levels between TS and NS. Correlation analysis showed that the average methylation level rather than a single site methylation level in #20 affected (P<0.05) mRNA expression. Collectively, it was found that the change of CD79B gene expression after MDV infection might be partly explained by modification of DNA methylation.

Quantitative proteomics identifies PTP1B as modulator of B cell antigen receptor signaling.

B cell antigen receptor (BCR) signaling is initiated by protein kinases and limited by counteracting phosphatases that currently are less well studied in their regulation of BCR signaling. Here, we used the B cell line Ramos to identify and quantify human B cell signaling components. Specifically, a protein tyrosine phosphatase profiling revealed a high expression of the protein tyrosine phosphatase 1B (PTP1B) in Ramos and human naïve B cells. The loss of PTP1B leads to increased B cell activation. Through substrate trapping in combination with quantitative mass spectrometry, we identified 22 putative substrates or interactors of PTP1B. We validated Igα, CD22, PLCγ1/2, CBL, BCAP, and APLP2 as specific substrates of PTP1B in Ramos B cells. The tyrosine kinase BTK and the two adaptor proteins GRB2 and VAV1 were identified as direct binding partners and potential substrates of PTP1B. We showed that PTP1B dephosphorylates the inhibitory receptor protein CD22 at phosphotyrosine 807. We conclude that PTP1B negatively modulates BCR signaling by dephosphorylating distinct phosphotyrosines in B cell-specific receptor proteins and various downstream signaling components.

Engineering an Antibody V Gene-Selective Vaccine.

The ligand-binding surface of the B cell receptor (BCR) is formed by encoded and non-encoded antigen complementarity determining regions (CDRs). Genetically reproducible or ‘public’ antibodies can arise when the encoded CDRs play deterministic roles in antigen recognition, notably within human broadly neutralizing antibodies against HIV and influenza virus. We sought to exploit this by engineering virus-like-particle (VLP) vaccines that harbor multivalent affinity against gene-encoded moieties of the BCR antigen binding site. As proof of concept, we deployed a library of RNA bacteriophage VLPs displaying random peptides to identify a multivalent antigen that selectively triggered germline BCRs using the human VH gene IGVH1-2*02. This VLP selectively primed IGHV1-2*02 BCRs that were present within a highly diversified germline antibody repertoire within humanized mice. Our approach thus provides methodology to generate antigens that engage specific BCR configurations of interest, in the absence of structure-based information.

HIgDFc-Ig inhibits B cell function by regulating the BCR-Syk-Btk-NF-κB signalling pathway in mice with collagen-induced arthritis

Rheumatoid arthritis (RA) is an autoimmune disease targeting the synovium. Previous studies have found that IgD may be a potential target for the treatment of RA. We designed a new type of fusion protein, hIgDFc-Ig (DG), to block the binding of IgD to IgD receptor (IgDR). In this study, we found that DG has a significant therapeutic effect in mice with collagen-induced arthritis (CIA). DG improved the claw of irritation symptoms in these mice, inhibited the pathological changes in spleen and joint tissues, and had a moderating effect on B cell subsets at different inflammatory stages. Moreover, DG could also decrease the levels of IgA, IgD, IgM and IgG subtypes of immunoglobulin in the serum of mice with CIA. In vitro, B cell antigen receptor (BCR) knockout Ramos cells were established using the CRISPR/Cas9 technology to further study the activation of BCR signalling by IgD and the effect of DG. We found that the therapeutic effect of DG in mice with CIA may be achieved by inhibiting the activation of BCR signalling by IgD, which may be related to the activation of Igβ. In summary, DG may be a potential biological agent for the treatment of RA and it has broad application prospects in the future.

Primary Immune Responses and Affinity Maturation Are Controlled by IgD.

Mature B cells co-express IgM and IgD B cell antigen receptors (BCR) on their surface. While IgM BCR expression is already essential at early stages of development, the role of the IgD-class BCR remains unclear as most B cell functions appeared unchanged in IgD-deficient mice. Here, we show that IgD-deficient mice have an accelerated rate of B cell responsiveness as they activate antibody production within 24h after immunization, whereas wildtype (WT) animals required 3 days to activate primary antibody responses. Strikingly, soluble monovalent antigen suppresses IgG antibody production induced by multivalent antigen in WT mice. In contrast, IgD-deficient mice were not able to modulate IgG responses suggesting that IgD controls the activation rate of B cells and subsequent antibody production by sensing and distinguishing antigen-valences. Using an insulin-derived peptide we tested the role of IgD in autoimmunity. We show that primary autoreactive antibody responses are generated in WT and in IgD-deficient mice. However, insulin-specific autoantibodies were detected earlier and caused more severe symptoms of autoimmune diabetes in IgD-deficient mice as compared to WT mice. The rapid control of autoimmune diabetes in WT animals was associated with the generation of high-affinity IgM that protects insulin from autoimmune degradation. In IgD-deficient mice, however, the generation of high-affinity protective IgM is delayed resulting in prolonged autoimmune diabetes. Our data suggest that IgD is required for the transition from primary, highly autoreactive, to secondary antigen-specific antibody responses generated by affinity maturation.

BAFF signaling in health and disease.

BAFF is a critical cytokine supporting the survival of mature naïve B cells, acting through the BAFFR receptor. Recent studies show that BAFF and BAFFR are also required for the survival of memory B cells, autoimmune B cells as well as malignant chronic lymphocytic leukaemia (CLL) cells. BAFFR cooperates with other receptors, notably the B cell antigen receptor (BCR), a process which is critical for the expansion of autoimmune and CLL cells. This crosstalk may be mediated by TRAF3 which interacts with BAFFR and with CD79A, a signalling subunit of the BCR and the downstream SYK kinase, inhibiting its activity. BAFF binding to BAFFR leads to degradation of TRAF3 which may relieve inhibition of SYK activity transducing signals to pathways required for B cell survival. BAFFR activates both canonical and non-canonical NF-κB signalling and both pathways play important roles in the survival of B cells and CLL cells.

The B-cell Receptor Autoantigen LRPAP1 Can Replace Variable Antibody Regions to Target Mantle Cell Lymphoma Cells.

Mantle cell lymphoma (MCL) accounts for 5%–10% of all lymphomas. The disease’s genetic hallmark is the t(11; 14)(q13; q32) translocation. In younger patients, the first-line treatment is chemoimmunotherapy followed by autologous stem cell transplantation. Upon disease progression, novel and targeted agents such as the BTK inhibitor ibrutinib, the BCL-2 inhibitor venetoclax, or the combination of both are increasingly used, but even after allogeneic stem cell transplantation or CAR T-cell therapy, MCL remains incurable for most patients. Chronic antigenic stimulation of the B-cell receptor (BCR) is thought to be essential for the pathogenesis of many B-cell lymphomas. LRPAP1 has been identified as the autoantigenic BCR target in about 1/3 of all MCLs. Thus, LRPAP1 could be used to target MCL cells, however, there is currently no optimal therapeutic format to integrate LRPAP1. We have therefore integrated LRPAP1 into a concept termed BAR, for B-cell receptor antigens for reverse targeting. A bispecific BAR body was synthesized consisting of the lymphoma-BCR binding epitope of LRPAP1 and a single chain fragment targeting CD3 or CD16 to recruit/engage T or NK cells. In addition, a BAR body consisting of an IgG1 antibody and the lymphoma-BCR binding epitope of LRPAP1 replacing the variable regions was synthesized. Both BAR bodies mediated highly specific cytotoxic effects against MCL cells in a dose-dependent manner at 1–20 µg/mL. In conclusion, LRPAP1 can substitute variable antibody regions in different formats to function in a new therapeutic approach to treat MCL.

SY-1530, a highly selective BTK inhibitor, effectively treats B-cell malignancies by blocking B-cell activation.

Objective:B-cell antigen receptor (BCR) signaling is required to maintain the physiological functions of normal B cells and plays an important pathogenic role in B-cell malignancies. Bruton tyrosine kinase (BTK), a critical mediator of BCR signaling, is an attractive target for the treatment of B-cell malignancies. This study aimed to identify a highly potent and selective BTK inhibitor.Methods:Homogeneous time-resolved fluorescence assays were used to screen BTK inhibitors. Typhoon fluorescence imaging and Western blot analysis were used to confirm the effects of SY-1530 on the BCR signaling pathway. Additionally, the anti-tumor activities of SY-1530 were evaluated in TMD8 xenografts and spontaneous canine B-cell lymphoma.Results:We found a novel irreversible and non-competitive inhibitor of BTK, SY-1530, which provided dose-dependent and time-dependent inhibition. SY-1530 selectively bound to BTK rather than inducible T-cell kinase; consequently, it did not significantly affect T-cell receptor signaling and caused limited off-target effects. SY-1530 blocked the BCR signaling pathway through down-regulation of BTK activity, thus leading to impaired phosphorylation of BTK and its downstream kinases. Moreover, SY-1530 induced apoptosis in a caspase-dependent manner and efficaciously inhibited tumor growth in mouse xenograft models of B-cell malignancy (P < 0.001). SY-1530 also induced positive clinical responses in spontaneous canine B-cell lymphoma.Conclusions:SY-1530 is an irreversible and selective BTK inhibitor that shows inhibitory effects on B-cell malignancies by blocking the BCR signaling pathway. Therefore, it may be a promising therapeutic approach for the treatment of B-cell malignancies.

The Actin-Disassembly Protein Glia Maturation Factor γ Enhances Actin Remodeling and B Cell Antigen Receptor Signaling at the Immune Synapse.

Signaling by the B cell antigen receptor (BCR) initiates actin remodeling. The assembly of branched actin networks that are nucleated by the Arp2/3 complex exert outward force on the plasma membrane, allowing B cells to form membrane protrusions that can scan the surface of antigen-presenting cells (APCs). The resulting Arp2/3 complex-dependent actin retrograde flow promotes the centripetal movement and progressive coalescence of BCR microclusters, which amplifies BCR signaling. Glia maturation factor γ (GMFγ) is an actin disassembly-protein that releases Arp2/3 complex-nucleated actin filaments from actin networks. By doing so, GMFγ could either oppose the actions of the Arp2/3 complex or support Arp2/3 complex-nucleated actin polymerization by contributing to the recycling of actin monomers and Arp2/3 complexes. We now show that reducing the levels of GMFγ in human B cell lines via transfection with a specific siRNA impairs the ability of B cells to spread on antigen-coated surfaces, decreases the velocity of actin retrograde flow, diminishes the coalescence of BCR microclusters into a central cluster at the B cell-APC contact site, and decreases APC-induced BCR signaling. These effects of depleting GMFγ are similar to what occurs when the Arp2/3 complex is inhibited. This suggests that GMFγ cooperates with the Arp2/3 complex to support BCR-induced actin remodeling and amplify BCR signaling at the immune synapse.

Integrating New Therapies for Chronic Lymphocytic Leukemia.

Over the last 2 decades, treatment of chronic lymphocytic leukemia (CLL) therapy has drastically changed, resulting in greatly improved survival and treatment tolerance with current targeted therapies. First, the transition from chemotherapy (alkylating agents, nucleoside analogs) to chemoimmunotherapy with the addition of anti-CD20 antibodies resulted in deeper and more complete remissions, with an improvement in progression-free and overall survival. Over the last few years, chemoimmunotherapy has gradually been replaced by new targeted agents, based on further improvement in survival, especially in patients with high-risk CLL, and fewer adverse effects, that is, a lack of myelosuppression and lack of DNA damage and associated risk of secondary acute myeloid leukemia/myelodysplastic syndrome. The most active targeted treatments for CLL patients are the kinase inhibitors, which inhibit signaling of surface receptors, especially the B-cell antigen receptor, and the BCL-2 antagonist venetoclax. Among the kinase inhibitors, Bruton tyrosine kinase inhibitors are highly effective and generally well-tolerated and induce durable responses in the vast majority of patients. PI3 kinase inhibitors are alternatives for patients with intolerance to Bruton tyrosine kinase inhibitors. This review discusses the rationale for the transition from chemotherapy-based treatment to the novel agents, the activity, adverse effects, sequencing, and combinations of the novel agents and provides an outlook into future CLL therapy for the next decade.

Cerdulatinib Pharmacodynamics and Relationships to Tumor Response Following Oral Dosing in Patients with Relapsed/Refractory B Cell Malignancies: Results from a Phase I Dose Escalation Study

Preclinical studies suggest that the spleen tyrosine kinase (SYK) and Janus kinase (JAK) family members can contribute to both tumor intrinsic and microenvironment-derived survival signals. A dual inhibitor of these kinases (cerdulatinib) was therefore developed, and a phase I dose-escalation study was completed to understand tolerability and efficacy, and their relationship to pharmacokinetics (PK) and pharmacodynamics (PD). Here, we report on PK/PD relationships and associations with tumor response in patients from the phase I dose-escalation portion of the clinical trial (NCT01994382).Repeat CT scans were obtained from 6 patients with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL), 12 patients with follicular lymphoma (FL), 12 patients with diffuse large B cell lymphoma, and 6 patients with mantle cell lymphoma. Meaningful clinical responses were observed in patients with CLL/SLL and FL, as previously reported (Hamlin et al, 2016); hence, the correlation between tumor response and PD markers was examined in these patient populations.Complete inhibition of SYK and JAK signaling pathways was achieved in whole blood of dosed patients at tolerated exposures. The level of target inhibition in blood correlated with serum cerdulatinib concentration, and the concentrations at which 50% inhibition was acheived (IC50) against B cell antigen receptor (BCR), interleukin (IL)-2, IL-4, and IL-6 signaling pathways ranged from 0.22-1.08 μM, depending on the phosphorylation event measured. Significant correlations were observed between inhibition of both SYK and JAK signaling pathways and tumor response, most notably the BCR and IL-4 signaling pathways. Serum markers of inflammation were also reduced following cerdulatinib administration, several of which significantly correlated with tumor response, including β-2 microglobulin, C-reactive protein, interferon γ-induced protein 10 (IP10), and macrophage inflammatory protein 3β (MIP3β). Of note, lymphocytosis was observed in CLL, SLL, and FL patients. Evidence of diminished tumor cell surface expression of CD69 and CD86, diminished CD5 expression (a negative regulator of BCR signaling), and enhanced C-X-C chemokine receptor type 4 (CXCR4) expression were observed in 2 patients with CLL, consistent with suppression of BCR and IL-4 signaling.Fresh tumor samples were obtained from the peripheral blood of 6 CLL patients treated with cerdulatinib, as well as archival tumor biopsies obtained from 4 FL patients and 1 MCL patient, which were submitted for next-generation sequencing. Clinical activity was observed in CLL patients bearing mutations to NOTCH1, ATM, p53, and KRAS, which are among the most frequently observed mutations in this disease. One of the responding patients bore a 17p deletion. Importantly, 2 patients with ibrutinib-relapsed CLL who progressed within the first cycle of therapy with cerdulatinib had 3 mutations in common: p53, EP300, and BTK (C481S). Genetic correlates in FL were more limited, with data on 4 patients, 3 of whom had evidence of tumor reduction and 1 which progressed. The 2 best responding patients with whom we have genetic information bore mutations to ZMYM3, KMT2D, FAT4, BCL2, BCL6, CD79B, and STAT6 . Lastly, strong clinical activity was observed in a transformed FL patient who presented with increased MYC, BCL2, and BCL6 expression by immunohistochemistry (“triple-hit” lymphoma).These data demonstrate the ability of cerdulatinib to potently and selectively inhibit SYK and JAK signaling at tolerated exposures in patients with relapsed/refractory B cell malignancies. Moreover, the extent of target inhibition, as well as suppression of inflammation, correlated with tumor response. Cerdulatinib is currently under investigation as part of a phase IIa study to further evaluate efficacy and tolerability in this population. DisclosuresCoffey:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership. Feng:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership. Betz:Portola Pharmaceuticals, Inc: Employment, Equity Ownership. Pandey:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership. Birrell:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership. Leeds:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership. Der:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership. Kadri:Portola Pharmacueticals, Inc.: Research Funding. Lu:Portola Pharmacueticals, Inc.: Research Funding. Segal:Portola Pharmacueticals, Inc.: Research Funding. Wang:Karyopharm: Research Funding; Portola: Research Funding. Michelson:Portola Pharmacueticals, Inc.: Employment. Curnutte:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership. Conley:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership, Patents & Royalties.

B-Cell Receptor Antigens As the Targeting Moiety for Bispecific Constructs - a Novel Treatment Approach for B-Cell Lymphomas

BackgroundChronic antigenic stimulation of the B-cell receptor (BCR) has been suggested to play a role in the pathogenesis of B-cell lymphomas, however only few B-cell receptor antigens have been identified until recently, when we identified ARS2 and LRPAP1 as the autoantigenic targets of the B-cell receptors from approximately 25% of DLBCLs of the ABC type and 45% of mantle cell lymphomas, respectively. These BCR antigens can be used to therapeutically target lymphoma cells in an approach we designated as BARs (B-cell receptor antigens for reverse targeting). To test whether BARs can substitute for the B-cell targeting antibody CD19, we designed T-cell engaging anti-CD3 single chain fragments (scFv) and NK-cell engaging anti-CD16 scFv with the respective BAR as the targeting moiety.Material and methodsVariable heavy- and light- chain genes of the anti-CD3 OKT3 hybridoma and the anti-CD16 3G8 hybridoma were cloned into a pcDNA 3.1 vector by standard cloning techniques, followed by DNA sequences of the autoantigens ARS2 or LRPAP1. VH and VL were linked by a glycine-serine linker, as was VL to the autoantigenic epitope resulting in a VH-(Gly₄Ser₁)ⁿ-VL-(Gly₄Ser₁)ⁿ-ARS2/LRPAP1 DNA sequence. The final cloning product was transfected in HEK 293 cells for production of bispecific constructs. Binding capacity to lymphoma cell lines (DLBCL: OCI-Ly3, U2932, HBL-1; MCL : MAVER-1, GRANTA-519) and PBMCs was assessed by flow cytometry. Western blot analysis was used for detection of bispecific constructs after incubation with the monoclonal anti-His Tag antibody. Cytotoxicity was evaluated by LDH release assay.ResultsWe cloned, expressed, characterized and tested 4 bispecific constructs: αCD3-ARS2, αCD3-LRPAP1, αCD16-ARS2 and αCD16-LRPAP1. αCD3 bispecific constructs bound specifically to CD3 on T cells whereas αCD16 constructs bound exclusively to CD16-positive cells. Bispecific ARS2 constructs bound specifically to DLBCL cells with a B-cell receptor specific for ARS2, while LRPAP1 containing constructs bound exclusively to MCL with a B-cell receptor specific for LRPAP1. αCD3-ARS2 and αCD3-LRPAP1 induced dose-dependent T-cell mediated, rapid cytotoxicity exclusively in lymphoma cell lines with B-cell receptors specific for ARS2 and LRPAP1, respectively, starting at concentrations as low as 0,1 µg/ml. NK-cell mediated cytotoxicity against the respective DLBCL and MCL cell lines was observed with αCD16 constructs αCD16-ARS2 and αCD16-LRPAP1 at concentrations starting from 1 µg/ml. Control lymphoma cell lines with BCR specificities other than for ARS2 or LRPAP1 were not affected.ConclusionBARs can substitute for B-cell-directed antibodies (e. g. CD19) as the targeting moiety in bispecific constructs for the therapeutic targeting of B-cell lymphomas. Because approaches using the BCR antigen for targeting the malignant B cells have an exclusive specificity for the cells with the specific surface BCR (which are exclusively the respective lymphoma cells), they do not only represent a sensu stricto tumor-specific approach, they can also be expected to be more effective (due to the higher density of BCR molecules on the lymphoma cell surface), but less toxic than the currently available bispecific T-cell and NK-cell engaging antibodies, because they leave all other cells, in particular normal B-lymphocytes, unaffected. Supported by Wilhelm-Sander-Stiftung DisclosuresThurner:Saarland university: Patents & Royalties: Saarland university has applied for a relevant patent. Regitz:Saarland university: Patents & Royalties: Saarland university has applied for a relevant patent. Fadle:Saarland university: Patents & Royalties: Saarland university has applied for a relevant patent.